Protein, Enzyme, and Biological Water Dynamics: 2D Vibrational Echo Spectroscopy

描述（由申请人提供）：研究建议研究一组相互关联的问题，涉及蛋白质，酶，酶-底物结合的动力学和动力学-结构关系，以及生物水的性质。该提案分为两个相关部分。第一部分讨论蛋白质和酶动力学。第二部分涉及生物水的性质及其对生物系统的影响。主要的实验工具是超快2D-IR振动回波光谱和其他超快IR方法。振动回波实验类似于2D-NMR，除了它们直接在其他方法无法访问的时间尺度上检查生物系统的结构/机械自由度。结合分子动力学模拟和其他理论方法对2D-IR结果进行了分析。在我们初步成功阐明底物结合和蛋白质动力学与2D-IR光谱之间的关系的基础上，新的方法将被应用于蛋白质结构动力学如何通过在活性位点中结合外源配体来修饰的重要问题。CO和叠氮化物探针将被选择性地引入几种过氧化物酶的活性位点内。关键结构基序和蛋白质功能之间的相互作用将被检查几个系统。脑红蛋白（Ngb）是一种血红素蛋白，具有一个二硫键，参与调节蛋白质氧结合亲和力。Ngb内发生的结构和动态转化将通过生物化学和致突变破坏二硫键来探测。蛋白质功能和结构转化之间的关系也将在其他系统，如nitrophorins检查。将采用最近开发的方法，在活性位点和蛋白质的特定位置选择性地引入蛋白质动力学的位点特异性探针。通过研究细胞色素c（cyt c）在水溶液和溶胶-凝胶纳米孔环境中的变性，探讨纳米限制对蛋白质去折叠的影响。用盐酸胍、尿素、甲醇和pH作为化学变性剂的变性研究将探测熔融球态的动力学性质。生物水与本体水行为显著不同，这是因为生物大分子受到纳米级限制和紧密接触的影响。我们成功的2D-IR测量纳米级水的动力学将扩展到反胶束与磷脂和非离子表面活性剂。水在蛋白质界面的动力学将通过将蛋白质限制在反胶束中并观察水氢键动力学来确定。由于水与跨膜蛋白和其他生物分子的相互作用，膜表面的水性质在生物过程中起着重要作用。将使用2D-IR光谱研究模型磷脂膜表面的水的动力学和相互作用。短杆菌肽，跨膜质子通道蛋白的模型，在多层中的水的动力学将通过超快红外光谱测定。复杂生物分子（如蛋白质和酶）的结构动力学决定了它们如何发挥其生物功能。该项目使用先进的红外激光技术直接研究生物分子结构动力学以及生物分子与周围介质（特别是生物环境中的水）的相互作用如何影响结构动力学。该方法建立在以前的成功应用和发展的最先进的超快红外激光实验。

DESCRIPTION (provided by applicant): Research is proposed to study an interrelated set of problems involving the dynamics and dynamics-structure relationships of proteins, enzymes, enzyme-substrate binding, and the nature of biological water. The proposal is organized into two related parts. The first part discusses protein and enzyme dynamics. The second part involves the properties of biological water and its impact on biological systems. The principal experimental tools are ultrafast 2D-IR vibrational echo spectroscopy and other ultrafast IR methods. The vibrational echo experiments are akin to 2D-NMR except that they directly examine the structural/mechanical degrees of freedom of biological systems on time scales not accessible by other methods. The 2D-IR results are analyzed in conjunction with molecular dynamics simulations and other theoretical approaches. Building on our initial successful work in elucidating the relationship between substrate binding and protein dynamics with 2D-IR spectroscopy, novel approaches will be applied to the important question of how protein structural dynamics are modified by binding of exogenous ligands in the active site. CO and azide probes will be introduced selectively within the active site of several peroxidases. The interplay between key structural motifs and protein function will be examined for several systems. Neuroglobin (Ngb) is a heme protein with a single disulfide bond that is implicated in modulating the protein oxygen binding affinity. Structural and dynamic transformations that occur within Ngb will be probed by biochemically and mutagenically disrupting the disulfide bond. The relationship between protein function and structural transformation will also be examined in other systems such as nitrophorins. Recently developed methodology to introduce site-specific probes of protein dynamics selectively within the active site and at specific locations in the protein will be employed. The effects of nanoscopic confinement on protein unfolding will be probed by studying the denaturation of cytochrome c (cyt c) in aqueous and sol-gel nanopore environments. Denaturation studies with guanidine HCl, urea, methanol, and pH as chemical denaturants will probe the dynamical properties of molten globule states. Biological water differs markedly from bulk water behavior because of the effects of nanoscopic confinement and intimate contact to biological macromolecules. Our successful 2D-IR measurements of the dynamics of nanoscopic water will be extended to reverse micelles with phospholipid and non-ionic surfactants. The dynamics of water at protein interfaces will be determined by confining proteins in the reverse micelles and observing the water hydrogen bond dynamics. Water properties at membrane surfaces play an important role in biological processes because of water's interaction with transmembrane proteins and other biomolecules. The dynamics and interactions of water at the surfaces of model phospholipids membranes will be studied using 2D-IR spectroscopy. The dynamics of water in gramicidin, a model for transmembrane proton channel proteins, in multibilayers will be determined via ultrafast IR spectroscopies. PUBLIC HEALTH RELEVANCE The structural dynamics of complex biological molecules, such as proteins and enzymes, determine how they perform their biological functions. This project is using advanced infrared laser techniques to directly examine biomolecular structural dynamics and how biomolecule interactions with the surrounding medium, particularly water in biological environments, influence structural dynamics. The methodology builds on previous successful applications and developments of state-of-the-art ultrafast infrared laser experiments.